How is a bacterial library created and a gene cloned? A bacterial library is constructed by chopping up the genome of the medicinal plant (the "parent") with a restriction enzyme. Restriction enzymes recognize specific double-stranded DNA sequences and cut each DNA strand, often creating "sticky ends" known as 3' or 5' overhangs. This generates a random assortment of DNA fragments. Some of these DNA fragments from the medicinal plant will contain the gene that encodes the green glowing protein with anticancer properties. When the same restriction enzyme is used to cut (digest) a plasmid (the "destination") it produces an opening in the plasmid with sticky ends that are complementary to the sticky ends of the medicinal plant DNA fragments. Next, the plant DNA fragments are inserted into plasmids by joining (ligating) their sticky ends and covalently bound together by a DNA ligase enzyme. The resulting plasmids contain random assortments of DNA fragments derived from the genome of the medicinal plant. The plasmids are transformed into the bacteria, E. coli. When transformed bacteria divide, the plasmid is also replicated. In the best-case scenario, some of the transformed bacterial cells will contain copies of the plasmid containing the plant gene, which encodes the protein of interest.

Secrets of the Rainforest colonies and GFP separation on a series of hydrophobic interaction chromatography (HIC) columns.

The Secrets of the Rainforest™ Kit provides a biotechnology curriculum that takes students on an adventure starting in the rainforest in the Andes, continuing to a biotechnology company engaged in developing new pharmaceutical compounds, and finally to the Food and Drug Administration.

In the hands-on portion of the exercise, students streak out the bacterial library, grow the bacteria, and select the colonies that have acquired the green glowing trait from a background of nonglowing bacteria. A culture of bacteria is grown to scale up production of the green glowing protein, which is then purified by column chromatography. The actual source of this fluorescent protein is the bioluminescent jellyfish Aequorea victoria.

This practical activity is followed by dry-lab lessons in which the purified green protein is put through the drug discovery process. This includes learning about Food and Drug Administration regulations, animal testing, marketing practices for introducing a new drug, financing, ethical issues, and student presentations. Students examine the needs and viewpoints of advocacy groups, the biotechnology industry, and the FDA. They consider ethical, economic, social, and practical issues, and develop possible strategies for what they feel are the best outcomes.

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Four-session laboratory activity, 45 min per session

Provides sufficient materials for eight student workstations, up to four students per workstation

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